The subcellular distribution of the NADPH oxidase of guinea-pig peritoneal-elicited macrophages was investigated. Post-nuclear supernatants obtained from PMA-stimulated macrophages were fractionated in discontinuous sucrose gradients. The NADPH oxidase was found to be enriched at the interface between 20 and 34 per cent sucrose. This interface was also enriched in 5'-nucleotidase, a plasma membrane marker and in glucose-6-phosphatase and NADPH-cytochrome c reductase, two endoplasmic reticulum markers. The distribution in the gradient of beta-glucuronidase, a marker of lysosomes and of succinate dehydrogenase, a marker of mitochondria was clearly different from that of NADPH oxidase and of the markers of plasma membrane and of endoplasmic reticulum. These results indicated that in stimulated-elicited macrophages the NADPH oxidase is associated with a membrane fraction. With the fractionation technique employed it was not possible to clarify whether the oxidase is located in the plasma membrane or in the endoplasmic reticulum. In order to clarify this matter the isolation of phagosomes was performed. NADPH oxidase was found to be enriched in the phagosomal fraction. Phagosomes were also found to be enriched in the plasma membrane marker 5'-nucleotidase. Glucose-6-phosphatase,, a marker of endoplasmic reticulum, and beta-glucuronidase, a marker of lysosomes were not enriched in the phagosomal fraction. The results obtained clearly suggest that the activated NADPH oxidase of peritoneal elicited macrophages of guinea pig is located in the plasma membrane.

The interaction of Escherichia coli 0111:B4 with polymorphonuclear leukocytes in the presence of specific antibodies and complement was studied. This strain, which is resistant to phagocytosis by polymorphonuclear leukocytes, may be ingested and killed by the phagocytes in the presence of both antibodies and fresh serum. The ineffectiveness of fresh serum to promote ingestion of E. coli 0111:B4 by the phagocytes in the absence of antibodies reflects the inability of this strain to activate the complement system through the alternative pathway. Investigation of the mechanisms of the bacterial killing by polymorphonuclear leukocytes showed that both antibodies and complement were required for the oxygen-independent bactericidal system, whereas they were not needed for the oxygen-dependent system.